Drug-resistant breast cancer cells are the main cause of recurrent disease. One mechanism of cancer cell survival and drug resistance is mobilization of GRP78/BiP, master-regulator of unfolded protein response and ER stress. Because of its role, GRP78/BiP is considered as a novel predictor of responsiveness to chemotherapy in breast cancer. We hypothesize that targeted destruction of GRP78/BiP in breast cancer cells would increase the efficacy of approved chemotherapeutic drugs. To test this hypothesis, we constructed a fusion protein comprising human epidermal growth factor (EGF) and SLiP, a catalytic subunit of a novel bacterial toxin SubAB, whose unique and the only substrate is GRP78/BiP. Our preliminary results indicate that in EGFR-positive cells, EGF-SLiP cleaves GRP78/BiP and inhibits cell growth at picomolar concentrations. Critically for this project, doxorubicin-resistant cancer cells display greater sensitivity to EGF-SLiP than parental cells. Since EGFR overexpression is also associated with increased drug resistance of breast cancer cells, we expect that targeting SLiP via EGFR would be especially beneficial. We also expect that the mechanism-based complementarity between EGF-SliP and chemotherapeutic drugs, as well as the absence of pre-existing immunity against SLiP, will provide EGF-SLiP with distinct advantages relative to previous unsuccessful EGF-based cytotoxins. In response to critique of the original proposal and in view of the new data, we redesigned the proposal and focused exclusively on animal studies in an orthotopic breast cancer model. The key questions regarding potential utility of EGF-SLiP for treatment of breast cancer will be explored in this project: 1) tumor penetration and GRP78/BiP cleavage, 2) systemic toxicity and immunogenicity, and 3) efficacy of combination with doxorubicin for treatment of doxorubicin- resistant tumors. In response to the critique of the resubmission we explain in more details the master regulator role of BiP which allows EGF-SLiP to act synergistically with various insults and add additional data. Successful completion of Phase I of this project will provide critical information on key EGF- SLiP characteristics in vivo. We expect that the results of Phase I will be sufficient for rational decision on feasibility of clinical development of EGF-SLiP. Phase II of this project will include side-by-side comparison with existing EGFR-directed drugs, formal toxicology, GMP- production, and transition to Phase I clinical trials. PUBLIC HEALTH RELEVANCE: We propose to test the feasibility of using a new cytotoxin, EGF-SLiP for targeting drug-resistant cells in the course of chemotherapy of breast cancer. The toxic part of the cytotoxin, SLiP, is a recently characterized bacterial protease that cleaves only one protein in mammalian cells, GRP78/BiP, which controls cellular defenses against chemotherapeutic drugs and is a novel predictor of resistance to chemotherapy of breast cancer. EGF-SLiP targets cytotoxin to breast cancer cells that express EGF receptors, a feature that is also implicated in cancer progression and drug resistance. Using orthotopic mouse model of breast cancer, we will establish efficacy of EGF-SLiP in cleaving GRP78/BiP in tumors, assess systemic toxicity and immunogenicity, and establish its efficacy as part of a combination regimen in treating doxorubicin-resistant tumor. We expect that this data establish feasibility of clinical development for EGF-SLiP. [unreadable] [unreadable] [unreadable]